US7901920B2 - Virosome-like-particles - Google Patents

Virosome-like-particles Download PDF

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US7901920B2
US7901920B2 US10/544,939 US54493904A US7901920B2 US 7901920 B2 US7901920 B2 US 7901920B2 US 54493904 A US54493904 A US 54493904A US 7901920 B2 US7901920 B2 US 7901920B2
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virus
virosome
short
membrane
particles
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Anke Luise Wilhelmine Huckriede
Jørgen Martin de Jonge
Antonius Johannes Hendrikus Stegmann
Pieter Joseph Schoen
Jan Christiaan Wilschut
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Bestewil Holding BV
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16123—Virus like particles [VLP]
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36123—Virus like particles [VLP]

Definitions

  • the invention relates to the production of virosome-like-particles.
  • Vaccines against membrane-containing (enveloped) viruses mostly consist of killed or live attenuated viruses, or a preparation of their proteins (e.g. split virus vaccines or subunit preparations). Vaccination with killed viruses and protein preparations is safer than vaccination with live attenuated viruses, because the latter may mutate or revert back to wild-type virus.
  • Subunit vaccines have the clear advantage that they can be prepared from viral proteins expressed by cells rather than from virus, making production safer and eliminating the risk of contaminating vaccine preparations with live viruses.
  • live viruses generally induces a strong immune response, protecting against future infections by the virus, protein preparations may fail to do so.
  • live attenuated viruses infect cells of the body, and will be replicated by these cells to some extent, after which the infected cells and viruses are detected by cells of the immune system, triggering an immune response.
  • Live or killed viruses can also be taken up by specialized phagocytic cells of the immune system, such as dendritic cells, and be presented to other cells of the immune system, triggering an immune responses. These phagocytic cells patrol the body, ingesting particles of the size of viruses all the time, but they do not efficiently take up the purified proteins of split virus or subunit vaccines [1-2].
  • ISCOMs are cage-like particles containing viral proteins complexed with adjuvants such as saponins like Quil A®), usually isolated from the bark of Quillaia sopanaria Molina.
  • adjuvants such as saponins like Quil A®
  • these adjuvants form the typical ISCOM structures of between 30-40 nm, rendering the antigen sufficiently particulate for uptake by phagocytic cells of the immune system, while acting at the same time as an adjuvant.
  • ISCOMs have been used in a number of veterinary vaccines, and strongly enhance the immunogenicity of the viral membrane proteins, the development of such vaccines for humans has been inhibited by concerns about their toxicity and the complexity of the mixture [3].
  • proteosomes (US application 0010053368) [4], consists of complexes of antigenic proteins such as the influenza hemagglutinin or the human immunodeficiency virus envelope glycoprotein, mixed with the purified outer membrane proteins of bacteria such as meningococci. While these multiple bacterial proteins may act as adjuvants, the complex nature of such mixtures, consisting of multiple proteins, lipids and other substances, will present a regulatory issue. Moreover, the immune response is directed against all proteins and other antigens present in the solution, and less specifically against the viral proteins.
  • virosomes are lipid bilayers containing viral glycoproteins derived from enveloped viruses.
  • Virosomes are generally produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent, followed by removal of this detergent from the extracted lipids and viral membrane proteins, in fact reconstituting or reforming the characteristic lipid bilayers (envelopes) that surround the viral core or nucleocapsid [5].
  • Influenza virus and Semliki Forest virus are two classical examples of enveloped viruses.
  • the first step in infection of cells by these viruses is uptake of intact viral particles by receptor-mediated endocytosis. Inside the endosomal compartment the conditions are mildly acidic due to the activity of a membranous ATP-dependent proton pump. Under these conditions (pH 5-6), the viral spike proteins undergo a conformational change which results in triggering of viral membrane fusion activity. Subsequent fusion of the viral membrane with that of the endosome results in cytoplasmic penetration of the viral genome, and the cell can be considered infected [6].
  • Enveloped viruses in general carry specific membrane proteins (the “spikes”) which are required for binding to and entry of cells.
  • influenza virus carries about 500 copies of hemagglutinin (HA), which is composed of two disulfide-linked subunits, HA1 and HA2, and which forms trimers in the viral membrane [7].
  • the HA1 subunits form the top domain of the spike and carry a pocket responsible for binding of the virus to its plasma membrane receptor, sialylated lipids (gangliosides) and proteins.
  • the stem region of the spike is mainly composed of the three HA2 subunits.
  • Each HA2 subunit contains a N-terminal fusion peptide, a highly conserved apolar sequence. Upon the conformational change induced by exposure to mildly acidic pH these peptides interact with the target membrane leading to fusion [6].
  • SFV and influenza enter cells through receptor-mediated endocytosis and fusion from within acidic endosomes, the molecular mechanisms of membrane fusion mediated by SFV and influenza virus are quite different.
  • Each Semliki Forest virion contains 80 spikes, which are each composed of three E1/E2 heterodimers [8]. These two membrane proteins have separate functions during the viral life cycle.
  • E2 is involved in virus-receptor binding
  • E1 mediates the merging of the viral and endosomal membranes.
  • E1/E2 complex dissociates and E1 rearranges to form homotrimers
  • influenza HA has a well-defined N-terminal fusion peptide, E1 does not.
  • Another prominent difference between both viruses is that HA-mediated fusion is not very sensitive to target membrane lipid composition [9]. Fusion of SFV has a strict requirement for the presence of cholesterol [11-12] and sphingolipid [13-17] in the target membrane.
  • virosomes An essential feature of virosome-like-particles obtained by reconstitution (herein also called virosomes) is that they are particles of the size that is efficiently taken up by phagocytic cells of the immune system, and they closely mimick the composition, surface architecture and functional activities of the native viral envelope. Virosomes that are particularly active in inducing an immune response were found to have maintained the proper functions of the envelope proteins of the native virus, such as membrane fusion, receptor-binding and other activities. Preservation of receptor-binding and membrane fusion activity is essential for expression of full immunogenic properties of said virosomes.
  • the viral membrane In the process used for the formation of virosomes, the viral membrane (envelope) is reformed during detergent removal. This step is thought to be necessary for a functional reconstitution of the native viral envelope, but quite difficult to control.
  • Current detergent removal protocols that result in reconstitution are mostly based on detergents with a low critical micelle concentration (cmc), and such detergents are particularly difficult to remove, in contrast to detergents with a comparatively high cmc, which can be removed by dialysis or ultrafiltration.
  • cmc critical micelle concentration
  • the latter type of detergent does not generally properly reconstitute viral membrane proteins, including the influenza virus hemagglutinin, leading predominantly to the formation of empty membranes on the one hand, and protein aggregates on the other.
  • a fluorescent lipid pyrene-labeled phosphatidylcholine (pyrPC)
  • pyrPC pyrene-labeled phosphatidylcholine
  • the invention provides a method for producing a virosome-like-particle comprising contacting an enveloped virus with a solution containing a short-chain phospholipid allowing solubilisation of the viral envelope of said virus further comprising removing the short-chain phospholipid from said solution allowing formation of a functionally reconstituted viral envelope.
  • the method is based on the use of a short-chain lecithin (phosphatidylcholine), which is well removable by dialysis.
  • the membrane-fusion activities of the reconstituted viral envelopes (virosomes) corresponded well to the pH-dependent fusion characteristics of the intact viruses, and to those of virosomes prepared with C12E8.
  • Retroviridae such as Human Immunodeficiency virus (HIV); rubellavirus; paramyxoviridae such as parainfluenza viruses, measles, mumps, respiratory syncytial virus, human metapneumovirus; flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis, St.
  • Retroviridae such as Human Immunodeficiency virus (HIV); rubellavirus; paramyxoviridae such as parainfluenza viruses, measles, mumps, respiratory syncytial virus, human metapneumovirus; flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis, St.
  • Herpesviridae such as Herpes Simplex virus, cytomegalovirus, Epstein-Barr virus; Bunyaviridae; Arenaviridae; Hantaviridae such as Hantaan; Coronaviridae; Papovaviridae such as human Papillomavirus; Rhabdoviridae such as rabies virus.
  • Coronaviridae such as human coronavirus; Alphaviridae, Arteriviridae, filoviridae such as Ebolavirus, Arenaviridae, poxyiridae such as smallpox virus, or African Swine Fever virus, in the method as provided herein.
  • the major membrane proteins of enveloped viruses are efficiently solubilised and reconstituted with a short-chain phospholipid, as long as it has a critical micelle concentratin (CMC) that is higher than 0.1 mM, such as with 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC) or 1,2-dicaproyl-sn-phosphatidylcholine (DCPC).
  • CMC critical micelle concentratin
  • DHPC 1,2-diheptanoyl-sn-phosphatidylcholine
  • DCPC 1,2-dicaproyl-sn-phosphatidylcholine
  • the membrane fusion activities of the reconstituted viral envelopes corresponded well to the pH-dependent fusion characteristics of the intact viruses, showing that short-chain phospholipids functionally reconstitute envelope proteins such as HA and E1/E2.
  • virosome-like-particles also appear comparable to virosomes prepared with C12E8 by a method previously developed in our laboratory.
  • the membrane fusion activity demonstrated here was based on lipid mixing of the membrane fluorophore pyrPC. This fluorophore was incorporated into the virosomal membrane during reconstitution; other amphipathic molecules may be incorporated in the same manner.
  • the method further comprises removal of viral nucleocapsid from the virus solubilized by the short-chain phospholipid, e.g. by centrifugation.
  • Said short-chain phospholipid is preferably removed by dialysis or filtration.
  • said phospholipid is a short-chain phosphatidylcholine, e.g. with a critical micelle concentration (cmc) of larger than 0.1 mM, preferably larger than 1 mM.
  • Preferred short-chain phospholipids according to the invention are 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC) or 1,2-dicaproyl-sn-phosphatidylcholine.
  • DHPC is structurally a phospholipid, but its short fatty-acyl chains of seven carbon atoms render it more water-soluble than other phospholipids.
  • the cmc of such molecules increases as the length of the fatty-acyl chains decrease [24].
  • dinonanoyl-phosphatidylcholine has a cmc of about 0.03 mM
  • the cmc values of dicapryloyl-phosphatidylcholine, DHPC, and DCPC are about 0.3, 2 and 14 mM, respectively.
  • the invention provides a method further comprising addition of a molecule that is not derived from said virus to said virosome-like-particle.
  • a molecule may be delivered to the cell that takes up the virosome-like-particle, and may for example be a nucleic acid, a lipid or a protein, preferably derived from a pathogen, such as a virus, a bacterium or a parasite, or a tumor-specific molecule.
  • a pathogen such as a virus, a bacterium or a parasite, or a tumor-specific molecule.
  • the invention also provides a virosome-like-particle obtainable by a method as provided herein, in particular for the production of a pharmaceutical composition, such as a vaccine.
  • a pharmaceutical composition such as a vaccine is suitable for parenteral or mucosal application.
  • a pharmaceutical composition comprising a virosome-like-particle according to the invention can also be used as delivery vehicle for nucleic acid or proteins. This novel method of virosome preparation by gentle dialysis-mediated phospholipid removal is especially of importance for the further use of virosome-like-particles as fusogenic carrier systems.
  • FIG. 1 Sucrose gradient analysis of influenza virosome-like particles
  • FIG. 2 Electron micrograph of influenza virosome-like particle.
  • Negative stain (ammonium molybdate) electron micrograph of influenza virosome-like-particles; bar 100 nm.
  • FIG. 3 DCPC-mediated solubilisation of Semliki Forest virus
  • Virus was mixed with DCPC, and subjected to ultracentrifugation. Aliquots of the unseparated virus (V), the supernatant (S) and the pellet (P) were subjected to polyacrylamide gelelectrophoresis in the presence of SDS. Protein bands corresponding to the E1 and E2 spike proteins and the capsid protein (C) are indicated.
  • FIG. 4 Rates of fusion of intact influenza virus ( ⁇ ) and of DCPC-reconstituted influenza virus envelopes ( ⁇ ) with erythrocyte target membranes
  • R18-labeled virus or pyrPC-labeled DCPC-reconstituted envelopes were mixed with erythrocyte ghosts at neutral pH at 37° C. At time 0 the medium was acidified to the pH value indicated. Fusion was continuously monitored by following R18 dequenching or decrease of pyrene excimer fluorescence. Rates of fusion were obtained from the fusion curves by determining the slopes of the tangents to the initial parts of the curves.
  • FIG. 5 Fusion of Semliki Forest DCPC virosomes with target liposomes
  • Reconstituted envelopes were mixed with target liposomes at neutral pH (pH 7.4) at 37° C. At time 0 the medium was acidified to the pH values indicated. Fusion was continuously monitored by following the decrease of pyrene excimer fluorescence.
  • the present invention provides an alternative method allowing upscaling of virosome-like-particle production. Instead of using a detergent to solubilize the viral membrane and detergent removal with a hydrophobic resin to reform the envelope of a virus preparation, a short-chain phospholipid is used in these steps, facilitating production of functionally reconstituted virosome-like-particles at an industrial scale.
  • Virosome-like-particles are produced by solubilization of viral membranes by short-chain phospholipids and purification of the viral membrane components, followed by removal of the short-chain phospholipids.
  • Short-chain phospholipids contain acyl chains with less than twelve carbon atoms each.
  • said short-chain phospholipid is a phosphatidylcholine.
  • said short-chain phospholipid is 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC) or 1,2-dicaproyl-sn-phosphatidylcholine (DCPC).
  • said short-chain phospholipid is produced synthetically or semi-synthetically.
  • the virosome-like-particles of the present invention can be applied for vaccination with antigens that would not normally elicit a sufficient immune response, if they were not present in the membrane of virosome-like-particles.
  • a sufficient immune response in the treated subject is a response that protects against subsequent infection by the pathogenic organism comprising the antigen.
  • the antigens that are part of the virosome according to the invention should have a hydrophobic part that is embedded in the lipid bilayer membrane of the virosome particle.
  • Many pathogenic entities such as viruses, bacteria, yeasts and parasites carry in their capsid, cell wall or membrane, proteins that elicit an immune response in the host.
  • antigens that have hydrophobic elements and that are suited to be part of a virosome according to the invention are proteins present in the membrane (also called envelope in the case of viruses) of the pathogen. Therefore, in one embodiment, the antigen present in the virosome of the invention is an integral membrane protein.
  • said antigens are derived from a virus, a parasite, a bacterium or a tumor cell. Especially preferred are virosome-like-particles, wherein said antigen is derived from influenza virus or an alphavirus such as Semliki Forest virus (SFV).
  • SFV Semliki Forest virus
  • Proteins from influenza virus that can be used in virosome-like-particles of the present invention are preferably the hemagglutinin (HA) protein, the neuraminidase (NA) protein and/or the M2 protein, alone or in combination, and those from SFV that can be applied are the spike proteins E1 and E2, preferably in combination.
  • Viruses that can be applied and used in the formation of the virosome-like-particles according to the invention can be derived from all sorts of viruses, non-limiting examples of such viruses being: Retroviridae such as Human Immunodeficiency virus (HIV); rubellavirus; paramyxoviridae such as parainfluenza viruses, measles, mumps, respiratory syncytial virus, human metapneumovirus; flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis, St.
  • Retroviridae such as Human Immunodeficiency virus (HIV); rubellavirus
  • paramyxoviridae such as parainfluenza viruses, measles, mumps, respiratory syncytial virus, human metapneumovirus
  • flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV
  • Herpesviridae such as Herpes Simplex virus, cytomegalovirus, Epstein-Barr virus; Bunyaviridae; Arenaviridae; Hantaviridae such as Hantaan; Coronaviridae; Papovaviridae such as human Papillomavirus; Rhabdoviridae such as rabies virus.
  • Coronaviridae such as human coronavirus; Alphaviridae, Arteriviridae, filoviridae such as Ebolavirus, Arenaviridae, poxyiridae such as smallpox virus, and African Swine Fever virus.
  • tumor-specific proteins are found to be proper entities that can be targeted by human or humanized antibodies. Such tumor-specific proteins are also within the scope of the present invention. Many tumor-specific antigens are known in the art. Therefore, in one preferred embodiment, the present invention provides virosome-like-particles comprising a tumor-specific antigen.
  • the present invention provides a method for producing a virosome, comprising some or all of the following steps: i) dissolving the virus in a short-chain phospholipid ii) removing the viral genetic material and core proteins and iii) removing the short-chain phospholipid under conditions that allow reformation of the membrane.
  • the method for producing a virosome disclosed by the present invention also comprises the step of purifying said virosome.
  • the present invention provides a pharmaceutical preparation comprising a virosome according to the invention, and a therapeutically acceptable carrier.
  • Therapeutically acceptable carriers for delivery are exemplified by water, buffered saline solutions, glycerin, and polysorbate 20, and may be buffered to provide a neutral pH environment.
  • the present invention provides the use of a virosome according to the invention, or a pharmaceutical preparation according to the invention, in therapy, prophylaxis or diagnosis.
  • the pharmaceutical preparation according to the invention is used for parenteral delivery.
  • the pharmaceutical preparation according to the invention is used for oral delivery, and in yet another preferred embodiment, the pharmaceutical preparation according to the invention is used for intranasal delivery. Stimulation of the immune system by these antigen-presenting virosome-like-particles may be due to a combination of their specific recognition by cells of the immune system, their particular character, the presentation of the protein, and the uncovering of hidden epitopes.
  • antigens proteins, peptides or polypeptides that have at least one hydrophilic and at least one hydrophobic moiety and that can elicit an immune response in the host whereto it is delivered.
  • integral membrane proteins are membrane proteins from tumor cells, from bacteria, parasites, yeasts and the envelope of viruses.
  • Viral membrane proteins that can be used can be derived from a wide range of viruses.
  • Retroviridae such as Human Immunodeficiency virus (HIV); a rubellavirus; paramyxoviridae such as parainfluenza viruses, measles mumps, respiratory syncytial virus, human metapneumovirus; flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis, St.
  • Retroviridae such as Human Immunodeficiency virus (HIV); a rubellavirus
  • paramyxoviridae such as parainfluenza viruses, measles mumps, respiratory syncytial virus, human metapneumovirus
  • flaviviridae such as yellow fever virus, dengue virus, Hepatitis C Virus (HCV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis, St.
  • HCV Hepatitis C Virus
  • JEV Japanese Encephalitis Virus
  • Herpesviridae such as Herpes Simplex virus, cytomegalovirus, Epstein-Barr virus; Bunyaviridae; Arenaviridae; Hantaviridae such as Hantaan; Coronaviridae; Poapavaviridae such as human Papillomavirus; Rhabdoviridae such as rabies virus; Coronaviridae such as human coronavirus; a Alphaviridae, Arteriviridae, filoviridae such as Ebolavirus, Arenaviridae, poxyiridae such as smallpox virus, and African Swine Fever virus.
  • Herpesviridae such as Herpes Simplex virus, cytomegalovirus, Epstein-Barr virus; Bunyaviridae; Arenaviridae; Hantaviridae such as Hantaan; Coronaviridae; Poapavaviridae such as human Papillomavirus; Rhabdoviridae such as
  • Influenza virus was produced by growing virus acquired from the World Influenza Center or the American Type Tissue Culture Collection (ATCC, Rockville, Md.), using methods known to persons skilled in the art, for instance by growing the virus on embryonated eggs or in cultured cells such as PER.C6TM or MDCK cells. The virus was then purified, by density gradient ultracentrifugation, and may subsequently be inactivated by treatment with beta-propiolactone or formaldehyde according to established procedures. Inactivation is not required for this procedure, and may affect the immune response.
  • the virus may be further purified by the resuspension of sedimented virus in 5.0 mM Hepes (pH 7.4), containing 0.15 mM NaCl and 0.1 mM EDTA (HNE-buffer), and loading this preparation atop linear 10-60% (w/v) sucrose gradients in HNE, centrifuging at 90,000 ⁇ g for 36 hr at 4° C. Virus can be pooled and aliquots stored at ⁇ 80° C. This additional purification step was not required for the formation of virosome-like-particles.
  • the efficiency of solubilization of the viral membrane was determined by analysis of the supernatant.
  • the amount of soluble protein present in the supernatant was found to correspond closely to the amount of hemagglutinin (HA) protein, the major membrane protein, that was present in the virus.
  • HA hemagglutinin
  • the supernatant was transferred to a 10,000 MW cut-off dialysis cassette (‘Slide-A-Lyzer’, Pierce Chemical Company, Rockford, Ill., U.S.A.) and dialyzed against 0.50 l HNE-buffer for 6 hr at 4° C., and subsequently against 0.50 l fresh HNE-buffer for 15 hr at 4° C.
  • ‘Slide-A-Lyzer’ Pierce Chemical Company, Rockford, Ill., U.S.A.
  • a sample of the dialyzed material was loaded on a linear gradient of 10-50% (w/v) sucrose in HNE-buffer and centrifuged for 24 hr at 100,000 ⁇ g at 4° C. ( FIG. 1 ). After centrifugation the gradient was fractionated, and the protein and phospholipid concentrations in the fractions were determined. The largest portions of phospholipid and protein were found in the bottom of the gradient in two fractions which contained about 40% and 45% sucrose, respectively. The 45% sucrose fraction contained 24% of the phospholipid and 38% of the protein recovered in the gradient and the 40% sucrose fraction contained 32% of the phospholipid and 46% of the recovered protein.
  • the formation of virosomes was confirmed by electron microscopy as shown in FIG. 2 .
  • the spikes can be clearly seen to protrude from the viral membrane.
  • Semliki Forest virus obtained from sources such as the ATCC, was produced on cell lines known in the art such as baby hamster kidney cells (BHK-21, from the ATCC) according to methods known in the art [23]. These cells were grown in Glasgow minimum essential medium, supplemented with 10% tryptose phosphate broth, 2.0 mM L-glutamine and 10% fetal bovine serum (all from GibcoBRL Life Technologies Inc., Paisley, UK). The medium was clarified by low-speed centrifugation (1,000 ⁇ g) at 4° C. for 10 min, and the virus was pelleted from clarified cell-culture medium by ultracentrifugation at 100,000 ⁇ g.
  • the virus was further purified by the resuspension of sedimented virus in 6.0 mM Hepes (pH 7.4), containing 0.15 mM NaCl and 0.1 mM EDTA (HNE-buffer), and loading this preparation atop linear 10-60% (w/v) sucrose gradients in HNE, centrifuging at 90,000 ⁇ g for 36 hr at 4° C. Virus was pooled and aliquots were stored at ⁇ 80° C. This additional purification step was not required for the formation of virosome-like-particles and does not affect the immune response.
  • the virus (0.25 ⁇ mol of phospholipid) was first sedimented by ultracentrifugation, and subsequently resuspended with 0.50 ml HNE-buffer, containing 100 mM DCPC. After a 30 min incubation at 0° C., the viral membrane was solubilized, and the nucleocapsids were removed by centrifugation for 20 min at 100,000 ⁇ g at 4° C. Efficient solubilization of the virus was demonstrated by an analysis of the supernatant and pellet by SDS-PAGE chromatography as shown in FIG. 3 .
  • the supernatant was transferred to a 10,000 MW cut-off dialysis cassette (‘Slide-A-Lyzer’, Pierce Chemical Company, Rockford, Ill., U.S.A.) and dialyzed against 0.50 l HNE-buffer for 6 hr at 4° C., and subsequently against 0.501 fresh HNE-buffer for 15 hr at 4° C.
  • ‘Slide-A-Lyzer’ Pierce Chemical Company, Rockford, Ill., U.S.A.
  • the X-47 strain of influenza virus (0.25 ⁇ mol phospholipid) was first sedimented by ultracentrifugation, and subsequently resuspended with 0.55 ml HNE-buffer, containing 25 mM DCPC. After a 30 min incubation at 0° C., the viral membrane was solubilized, and the nucleocapsids were removed by centrifugation for 20 min at 100,000 ⁇ g at 4° C. The supernatant was then added to a dried thin film of 10 nmol of (1-pyrenedecanoyl)-sn-phosphatidylcholine (pyrPC) (Molecular Probes, Leiden, The Netherlands).
  • pyrPC (1-pyrenedecanoyl)-sn-phosphatidylcholine
  • virosome-like-particles were then formed from this mixture by dialysis as described above.
  • the resulting virosome-like-particles were mixed with erythrocyte ghosts, prepared as described previously [25] from human red blood cells, at neutral pH (pH 7.4) at 37° C.
  • the medium was acidified as indicated. Fusion was monitored continuously by following the decrease of the pyrene excimer fluorescence.
  • a virus sample was labeled with octadecylrhodamine (R18) as previously described [27].
  • R18 octadecylrhodamine
  • SFV (1-pyrenedecanoyl)-sn-phosphatidylcholine
  • Liposomes used as targets in the fusion assay were prepared from egg phosphatidylcholine (PC), phosphatidylethanolamine (PE) derived from egg PC, bovine brain SPM and cholesterol (molar ratio 1:1:1:1.5) by a freeze-and-thaw/extrusion procedure [44].
  • PC egg phosphatidylcholine
  • PE phosphatidylethanolamine
  • cholesterol molar ratio 1:1:1:1.5
  • mice Immunization of mice by parenteral application of virosome-like particles reconstituted by the short-chain phospholipid method was compared to immunization with a conventional influenza subunit vaccine formulation.
  • Balb/C mice (5 animals per group) were immunized, on days 0 and 14, by intramuscular injection of 30-50 microliters of virosome-like preparation in HNE-buffer preparation, containing 5 ⁇ g of HA, derived from inactivated influenza virus (strain X-47H 3 N 2 ). Blood was collected on day 21. Serum samples were frozen in liquid nitrogen and stored at ⁇ 20° C. until analysis. Hemagglutination-inhibition (HI) titers were determined using guinea pig erythrocytes according to standard methodology. The results are described in Table I. It is shown that virosome-like-particles elicit a more potent virus-neutralizing antibody response than subunit antigen.
  • HNE-buffer preparation containing 5 ⁇ g of HA

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US20110281354A1 (en) * 2008-11-06 2011-11-17 Phaserx, Inc. Bispecific intracellular delivery vehicles
US8822213B2 (en) * 2008-11-06 2014-09-02 University Of Washington Bispecific intracellular delivery vehicles
US9220791B2 (en) 2008-11-06 2015-12-29 University Of Washington Bispecific intracellular delivery vehicles
WO2015000831A1 (en) 2013-07-02 2015-01-08 Crucell Holland B.V. Method for preparing virosomes
WO2015000832A1 (en) 2013-07-02 2015-01-08 Crucell Holland B.V. Method for preparing virosomes
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